The present invention relates to cells derived from delayed blastocysts and to cell lines generated therefrom.
Embryonic development starts soon after fertilization with blastomer cleavage, proliferation and differentiation. The blastomers within the developing mammalian embryo remain totipotent until the morula compaction stage. In the compacted embryo, the blastomers initiate polarization which results in two distinct cell-populations; the inner cell mass (ICM), which contributes to the embryo, and the outer trophectoderm layer, which develops into the extra embryonic layers. It is at this stage of embryogenesis—towards the end of the first week of development that embryonic stem (ES) cells are traditionally derived from the inner cell mass of the blastocyst.
At the time of implantation, the ICM is separated into a layer of primitive endoderm, which gives rise to the extra embryonic endoderm, and a layer of primitive ectoderm, which gives rise to the embryo proper and to some extra embryonic derivatives [Gardner J. Embryological Experiment and Morphology, 1982; 68: 175-198]. During the next major phase of development, termed gastrulation, the embryonic ectoderm differentiates into the three primary germ layers—endoderm (inside layer), mesoderm (middle layer), and ectoderm (outer layer). The cells become progressively restricted to a specific lineage, losing their pluripotency and thus are regarded as multi-potent progenitor cells. Therefore, pluripotent embryonic stem cells proliferate and replicate in the intact embryo only for a limited period of time.
Embryonic stem cells are characterized by their ability to propagate indefinitely in culture, as undifferentiated cells, while they can be induced to differentiate in vivo into teratomas when injected into SClD mice [Thomson, J. A., et al., (1998), Science, 282, 1145-1147; Reubinoff, B. E., et al., (2000), Nature Biotechnol., 18, 399-404]. They may also differentiate in vitro into embryoid bodies (EBs) that contain embryonic cells from the three germ layers (endoderm, mesoderm, and ectoderm). Moreover, this differentiation can be somewhat directed by the addition of growth factors into the culture media.
Human ES cells may also be genetically manipulated in culture [Eiges et al., (2001) Curr. Biol., 11, 514-518] and the transfected cells remain pluripotent and retain a normal karyotype [Shuldiner et al., (2003) Stem Cells 21, 257-265].
As a result of their unique features, it has been suggested that human ES cells hold the promise of changing the face of cell transplantation, by replacing or restoring tissue that has been damaged by disease or injury. Replacement of non-functional cells using ES cells technology can offer a lifelong treatment. Thus, diseases that might be treated by transplanting human ES-derived cells include Parkinson's disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart failure, and osteogenesis imperfecta.
Many other potential uses of human ES cells have been proposed that do not involve transplantation. For example, human ES cells could be used to study early events in human development. Human ES cells could also be used to test candidate therapeutic drugs or potential toxins by directing their differentiation into specific cell types. ES-derived cells may be more likely to mimic the in vivo response to the drug(s) being tested than animal and other in-vitro models and so offer safer, and potentially cheaper, models for drug screening.
Finally, human ES cells could be used to develop new methods for genetic engineering. Currently, the genetic complement of mouse ES cells in vitro can be modified easily by techniques such as homologous recombination. Using this method, genes to direct differentiation to a specific cell type or genes that express a desired protein product might be introduced into the ES cell line. Ultimately, if such techniques could be developed using human ES cells, it may be possible to devise better methods for gene therapy.
At present the only source for embryonic stem cells is the pre-implantation blastocyst embryo.
The pluripotency of human post-implantation embryonic cells between the time of implantation and the gastrulation process has as yet never been examined. Surani and Edwards teach in vitro culturing techniques of human embryos to day 9, demonstrating the presence of proliferating and healthy ICM [Edwards R. G., Surani M. A. H. (1978) Upsala Journal of Medical Sciences 22: 39-50].
However, they did not examine the pluripotency of stem cells at this post-implantation stage and did not isolate or culture them to allow their characterization.
Rathjen and colleagues teach a method for homogenous differentiation of mouse embryonic stem cells into early primitive ectoderm-like (EPL) using conditioned medium of Hep G2 cells [Rathjen et al. 1999, J. of Cells Science 112, 601-612] and demonstrate some similarities between them and the embryonic stem cells of the present invention. For example both cell types have a colony morphology of epithelial-like structures, a higher tendency to differentiate into mesodermal tissues and a reduced ability to either integrate into the embryonic germ layers after injection into mouse blastocysts [Lake et al. 2000, J. of Cells Science 113, 555-566] or form teratomas. However, in sharp contrast to the embryonic stem cells of the present invention, the EPLs unique features together with their ability to be cultured for limited passages in vitro are irreversible when the Hep G2 conditioned medium is removed [Rathjen et al. 1999 J. of Cells Science 112, 601-612; Lake et al. 2000, J. of Cells Science 113, 555-566]. The gene expression pattern of these cells is also different. The EPL cells, for example, express brachyury only as early EBs and not as undifferentiated cells like the embryonic stem cells of the present invention [Rathjen et al., 2000 J Cell Sci. 2000 113:555-66]. Together with the fact that the isolated EPLs are non-primate cells, the above mentioned differences indicate distinct cell populations.
The very broad range of potential applications for embryonic stem cells suggests that the identification of additional sources together with the novel stem cell lines derived therefrom, will be of critical importance for medical research in general and the advancement of stem cell research in particular.